Method of producing microbial transglutaminase

ABSTRACT

The present invention provides a neutral metalloprotease from actinomycetes which selectively cleaves a pro-structure part of a microbial protransglutaminase and a gene encoding said neutral metalloprotease. An active microbial transglutaminase having the pro-structure part cleaved can be obtained by culturing a microorganism into which a gene encoding the neutral metalloprotease from actinomycetes according to the present invention has been introduced, where by producing the neutral metalloprotease from actinomycetes, and reacting it on a microbial protransglutaminase.

This application is a continuation under 35 U.S.C. §120 ofPCT/JP2004/002923, filed Mar. 5, 2004, the entirety of which isincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a protease which efficiently cleavesthe pro-structure part of pro-transglutaminase resulting in an activetransglutaminase, and to a nucleic acid encoding the protease, whereinthe pro-transglutaminase is produced by actinomycetes. The presentinvention also relates to a method of producing microbialtransglutaminase in its active form using the protease. Additionally,the present invention relates to a method of producing a neutralmetalloprotease.

2. Brief Description of the Related Art

Transglutaminase is an enzyme which catalyzes the acyltransfer reactionof γ-carboxylamide groups in the peptide chain of the protein. When theenzyme reacts with a protein, the formation of the cross-linkageε-(γ-Glu)-Lys, and the replacement of Gln with Glu by deamidation canoccur. Transglutaminases have been used to manufacture gelled foodproducts such as jelly, yogurt, cheese, or gelled cosmetics and others,and to improve the quality of meat, etc. (Japanese publication ofexamined application (JP-Kokoku) No. 1-50382). Moreover,transglutaminase is highly useful in industry in that it has been usedto manufacture materials for thermostable microcapsules, carriers forimmobilized enzyme, etc.

Expression of animal transglutaminase activity is calcium-dependent, andtransglutaminases from microorganisms (microbial transglutaminase(s),which is/are also referred to as “MTG(s)” hereinafter), have beenpreviously reported to also be calcium-independent. A microbialtransglutaminase from a bacterium belonging to genus Streptoverticilliumhas been reported. Such Streptoverticillium bacteria include, forexample, Streptoverticillium griseocarneum IFO 12776,Streptoverticillium cinnamoneum sub sp. cinnamoneum IFO 12852,Streptoverticillium mobaraense (hereinafter, S. mobaraense) IFO 13819,and others (Publication of unexamined Japanese patent application(JP-Kokai) No. 64-27471).

Because these transglutaminases, however, have been produced viapurification from cultures of the microorganisms described above,problems have been reported regarding the produced amount, productionefficiency, and the like. Then, in an attempt to more efficientlysecrete heterologous proteins, a method was reported using a coryneformbacterium as a host, and a fused protein whereby transglutaminase wasconnected downstream of the signal peptide domain of the coryneformbacterium, and the transglutaminase was efficiently secreted resultingin a high yield of transglutaminase (WO 01/23591). In this study, amethod is also described wherein inactive MTG is secreted aspro-transglutaminase (referred to as “pro-MTG” hereinafter) whereby apro-structure part is connected to MTG, and then this pro-structure partis cleaved by a protease to convert it into an active transglutaminase.A further method is described wherein an active transglutaminase isdirectly produced in a culture medium by co-expressing SAM-P45, which isa serine protease derived from actinomycetes, in a sufficient amount ina coryneform bacterium which also produces the pro-MTG.

Although a method in which an active transglutaminase is directlyproduced by co-expressing pro-MTG and a protease which allows cleavageof the pro-structure part of the pro-MTG in a coryneform bacterium isassumed to be an extremely efficient method of producingtransglutaminase, the substrate specificity of SAM-P45 is not verystrict, and it may digest and degrade not only the pro-structure part ofthe pro-MTG but also the transglutaminase itself to some degree.Therefore, the handling of SAM-P45 may not be easy. When SAM-P45 isused, therefore, the production method of transglutaminase should bestrictly controlled such that degradation of the transglutaminase in theculture medium will not occur.

There is still, therefore, demand for a protease which can selectivelycleave only the pro-structure part of pro-MTG, with as littledegradation of the transglutaminase itself as possible during theproduction of an active transglutaminase.

A dispase derived from Bacillus polymyxa is known (Eur. J. Biochem.,vol. 257, p. 570-576 (1998)) to be an enzyme besides SAM-P45 whichcleaves the pro-structure part of pro-MTG. A large amount of the enzyme,however, is required to cleave the pro-structure part, and there is arisk of degrading the transglutaminase itself. In addition, dispase is areagent in cell culture, so it is expensive when used an enzyme forindustrial use.

SUMMARY OF THE INVENTION

There remains a need for a protease which can selectively cleave solelythe pro-structure part of pro-MTG, and degrade the transglutaminaseitself as little as possible during the production of an activetransglutaminase, as mentioned above. Additionally, if proteases whichselectively cleave only the pro-structure part of pro-MTG could be used,and thereby cause as little degradation as possible of thetransglutaminase itself, it would be advantageous for the production ofan active transglutaminase. Furthermore, if proteases for production oftransglutaminase which selectively cleave the pro-structure part ofpro-MTG were able to be secreted extracellularly, it would be morepreferable because active transglutaminases could be directly producedin the culture medium by co-expressing them with the pro-MTG.

It is an object of the present invention to provide a method ofproducing an active transglutaminase from a microbialprotransglutaminase comprising contacting a neutral metalloprotease withthe protransglutaminase, wherein said neutral metalloprotease isproduced by culturing a microorganism into which a gene encoding theneutral metalloprotease from actinomycetes has been introduced, andrecovering an active microbial transglutaminase.

It is a further object of the present invention to provide the method asdescribed above, wherein said microorganism comprises a coryneformbacterium.

It is a further object of the present invention to provide the method asdescribed above, wherein said neutral metalloprotease from actinomycetescomprises characteristics selected from the group consisting of amolecular weight of about 35,000, an optimum pH of pH7.0, stablity at pHof pH4-10, an optimum temperature of about 45° C., stability below about50° C., and said metalloprotease is strongly inhibited by ethylenediamine tetraacetic acid, 1,10-phenanthroline, phosphoramidon, andStreptomyces subtilisin inhibitor (SSI) from actinomycetes.

It is a further object of the present invention to provide the method asdescribed above, wherein said neutral metalloprotease from actinomycetescomprises characteristics selected from the group consisting of amolecular weight of about 71,000, an optimum pH of 7.0, stablity at pHof 5-10, an optimum temperature of about 55° C., and saidmetalloprotease is strongly inhibited by ethylene diamine tetraaceticacid, 1,10-phenanthroline, phosphoramidon, dithiothreitol, andStreptomyces subtilisin inhibitor (SSI) derived from actinomycetes.

It is a further object of the present invention to provide a neutralmetalloprotease from actinomycetes comprising characteristics selectedfrom the group consisting of a molecular weight of about 35,000, anoptimum pH of pH7.0, stablity at pH of pH4-10, an optimum temperature ofabout 45° C., stability below about 50° C., and said metalloprotease isstrongly inhibited by ethylene diamine tetraacetic acid,1,10-phenanthroline, phosphoramidon, and Streptomyces subtilisininhibitor (SSI) from actinomycetes.

It is a further object of the present invention to provide a neutralmetalloprotease from actinomycetes comprising characteristics selectedfrom the group consisting of a molecular weight of about 71,000, anoptimum pH of 7.0, stablity at pH of 5-10, an optimum temperature ofabout 55° C., and said metalloprotease is strongly inhibited by ethylenediamine tetraacetic acid, 1,10-phenanthroline, phosphoramidon,dithiothreitol, and Streptomyces subtilisin inhibitor (SSI) derived fromactinomycetes.

It is a further object of the present invention to provide a nucleicacid molecule encoding said protease described above.

It is a further object of the present invention to provide a method ofproducing said protease comprising culturing a coryneform bacterium intowhich the nucleic acid molecule as described above has been introduced,and recovering said neutral metalloprotease which has been secretedextracellularly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph which shows the pH dependence of SVP35 and SVP70activity.

FIG. 2 is a graph which shows the pH stability of SVP35 and SVP70.

FIG. 3 is a graph which shows the temperature dependence of SVP35 andSVP70 activity.

FIG. 4 is a graph which shows the temperature stability of SVP35.

FIG. 5 depicts the inhibitory activities of various compounds to SVP35and SVP70 activity.

FIGS. 6 (A), (B), and (C) depict the sequential change of the conversionof pro-MTG to an active MTG by SVP35 (A), and SVP70 (B) fromStreptoverticillium mobaraense, and by neutral metalloprotease SGMPII(C) from Streptomyces griseus, relative to the change in the proteinamount. The lanes represents the time elapsed, as indicated by the “(h)”notation in the figure panels.

FIGS. 7 (A) and (B) are graphs which depict the time course oftransglutaminase activity, if a pro-MTG is reacted with SVP70 andSAM-P45, respectively. (A): SVP70 addition, ●: additional amount of1/200 relative to substrate, ▪: additional amount of 1/500 relative tosubstrate; (B): SAM-P45 addition, ♦: additional amount of 1/10 relativeto substrate, ▴: additional amount of 1/50 relative to substrate.

FIGS. 8 (A) and (B) are graphs which depict the time course of theamount of MTG protein, if a pro-MTG reacts with SVP70 and SAM-P45,respectively. (A): SVP70 addition, ●: additional amount of 1/200relative to substrate, ▪: additional amount of 1/500 relative tosubstrate; (B): SAM-P45 addition, ♦: additional amount of 1/10 relativeto substrate, ▴: additional amount of 1/50 relative to substrate.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention describes a protease which selectively cleaves thepro-structure part of pro-MTG, but degrades the transglutaminase itselfas little as possible, and the isolatation and purification of a neutralmetalloprotease having such a property. The present invention alsodescribes a DNA encoding said protease, its introduction into a hostcoryneform bacterium, and the successful secretory expression. Inaddition, the described enzyme was actually reacted with the pro-MTG tocleave the pro-structure part, and the active transglutaminase wasrecovered. The present invention describes the identification of neutralmetalloproteases derived from microorganisms from other sources whichhave an equivalent function, and which have been proven similarly usefulfor the production of an active MTG.

Namely, the present invention is a neutral metalloprotease fromactinomycetes which has high selectivity in cleaving the pro-structurepart of pro-MTG, and a nucleic acid molecule encoding it.

The present invention also encompasses a method of producing an activeMTG by cleaving a pro-structure part of a pro-MTG with a neutralmetalloprotease.

The present invention also encompasses a method of producing saidmetalloprotease by introducing a nucleic acid molecule which encodessaid neutral metalloprotease into a coryneform bacterium, culturing thiscoryneform bacterium, thereby allowing the expression of said neutralmetalloprotease, and recovering the extracellularly secretedmetalloprotease.

More specifically, the present invention encompasses a neutralmetalloprotease SVP35 from actinomycetes having the followingproperties:

1) Molecular weight: about 35,000 (as measured by SDS-PAGE)

2) Optimum pH: 6.0-8.0, more specifically 6.5-7.5, in particular around7.0

3) pH Stability: pH of 4-10

4) Optimum temperature: about 45° C.

5) Temperature stability: it is stable below about 50° C.

6) It is strongly inhibited by ethylene diamine tetraacetic acid,1,10-phenanthroline, and phosphoramidon which are metalloproteaseinhibitors, and by Streptomyces subtilisin inhibitor (SSI) fromactinomycetes.

The present invention also encompasses a neutral metalloprotease SVP70having the following properties:

1) Molecular weight: about 71,000 (as measured by SDS-PAGE)

2) Optimum pH: the range of 6.0-8.0, more specifically 6.5-7.5, inparticular around 7.0

3) pH Stability: pH of 5-10

4) Optimum temperature: the range of about 50° C.-55° C., in particulararound 55° C.

5) It undergoes a strong inhibitory action by ethylene diaminetetraacetic acid, 1,10-phenanthroline, and phosphoramidon which aremetalloprotease inhibitors, dithiothreitol which is a SH-reductant, andby Streptomyces subtilisin inhibitor (SSI) derived from actinomycetes.

The present invention also encompasses a nucleic acid molecule encodingsaid SVP35 or SVP70.

The present invention also encompasses a method of producing an activeMTG, comprising cleaving the pro-structure part of pro-MTG by said SVP35or SVP70.

Furthermore, the present invention is a method of producing SVP35 orSVP70, comprising introducing a nucleic acid molecule encoding saidSVP35 or SVP70 into a coryneform bacterium, culturing the coryneformbacterium into which said nucleic acid molecule has been introduced, andrecovering the extracellularly secreted SVP35 or SVP70.

In general, it is known that a secretory protein is translated as aprepeptide or a prepropeptide and thereafter its signal peptide (thepre-part) is cleaved resulting in a mature peptide or propeptide. Thepropeptide is then cleaved at the domain referred to as a pro-structure,resulting in a mature peptide. As used herein, the pro-structure part ofa secretory protein may be simply referred to as “pro-structure”. Inaddition, as used herein, “a signal sequence” refers to the sequencewhich is located at the N-terminus of a secretory protein precursor andwhich is not present in a naturally occurring mature protein. The phrase“a signal peptide” refers to the peptide which is cleaved from theprotein precursor. Generally, a signal sequence is cleaved by a proteasefollowing extracellular secretion.

As used herein, a protein which does not contain a signal peptide butdoes contain a pro-structure part may be referred to as a “proprotein”,for example “protransglutaminase” or “pro-MTG”. As used herein, thepro-structure part of a secretory protein may be simply referred to as“a pro-structure” or “a pro-structure part”, and these terms can hereinbe used interchangeably.

Among proteases which are assumed to be easily expressed in a coryneformbacterium, a protease having high specificity and selectivity for thesubstrate of interest was sought, i.e. a protease which selectivelycleaves the pro-structure part of a pro-MTG, with as little degradationas possible of the transglutaminase itself.

When MTG is secreted extracellularly by an actinomycetes, it had beenassumed to be secreted first as a pro-MTG, followed by the cleavage ofthe pro-structure part pro-MTG, resulting in an active MTG (Eur. J.Biochem., vol. 257, p. 570-576 (1998)). Accordingly, a protease thatcleaves the pro-structure part of a pro-MTG within MTG-producingactinomycetes was expected to be present. Since this protease isoriginally a protease that cleaves the pro-structure part, it isexpected that the protease has a high selectivity for substrates andcleaves only the pro-structure part, while acting on the MTG itself to alesser degree.

In addition, both a structural gene of a pro-MTG of actinomycetes and astructural gene of the protease SAM-P45 can be effectively expressed ina coryneform bacterium, and secreted extracellularly. Based on thisinformation, an investigation was conducted in order to find theprotease of interest from a MTG-producing bacterium which is anactinomycetes, and as a result, it was revealed that the MTG-producingstrain Streptoverticillium mobaraense had high cleavage selectivity forthe pro-structure part of the pro-MTG and produces new neutralmetalloproteases useful for the production of an active MTG. Theseneutral metalloproteases were isolated and purified, and theirenzymological properties were demonstrated. Furthermore, the amino acidsequences of the N-terminal parts of these metalloproteases weredetermined, and the genes encoding the metalloproteases were obtained.

In addition, the enzyme gene was introduced into a coryneform bacterium,allowing the expression of it in a system using a coryneform bacteriumas a host, and as a result, the enzyme was secreted extracellularly.Furthermore, the enzyme was practically contacted with a pro-MTG of apro-structure part, resulting in the cleavage of the pro-structure partto yield an active transglutaminase. Neutral metalloproteases frommicroorganisms from other sources having an equivalent function werealso found, and which have been found to be similarly useful for theproduction of an active MTG.

More specific embodiments of the present invention will be illustratedhereinafter.

The neutral metalloproteases according to the present invention can beprepared from the surfaces of a cultured actinomycetes or culturesupernatant of the actinomycetes, including Streptoverticilliummobaraense, Streptomyces griseus, Streptomyces coelicolor, etc.

Next, the newly found neutral metalloproteases of Streptoverticilliummobaraense IFO13819 are described.

The cultivation of a bacterium to obtain the neutral metalloproteaseaccording to the present invention, for example, an actinomycetes asdescribed above, can be carried out according to the methodsconventionally used for the cultivation of actinomycetes. Namely, acommon medium containing conventional carbon sources, nitrogen sources,inorganic ions, and others can be used as a medium for the culture.Glucose, starch, sucrose, and others can be used as the carbon sources.Peptone, yeast extract, meat extract, malt extract, ammonium salt, andothers are optionally used as the nitrogen sources, if necessary. Theculture may be conducted under aerobic conditions which areappropriately controlled within the pH range of between pH 5.0 and 8.5and the temperature range between 15° C. and 37° C. For the productionof the neutral metalloproteases according to the present invention, theculture is preferably continued so that the maximum amount of thedesired neutral metalloprotease may be achieved, and then it can beterminated. Although the suitable culture period depends on thetemperature, pH, and the type of medium, usually the period ispreferably about 1 to 12 days. After the culturing period, the culturemay be separated into the cells and the culture supernatant bycentrifugation and the like.

The new neutral metalloproteases according to the present invention canbe obtained from the culture supernatant and/or the recovered cells, inparticular, from the surface of the cells. To purify the enzyme, methodswhich are conventionally used for purifying an enzyme, for example, anammonium sulfate salting-out technique, gel filtration technique,ion-exchange chromatography, hydrophobic chromatography, and the likecan be adopted. The protease can be purified more efficiently using highperformance liquid chromatography (HPLC) etc. The enzyme activity of theneutral metalloprotease obtained in this way can be determined byreacting the enzyme with a peptide which contains a region connectingthe pro-part of a protransglutaminase and a mature transglutaminase, forexample, a synthetic peptide Gly-Pro-Ser-Phe-Arg-Ala-Pro-Asp-Ser (SEQ IDNO: 11) (Peptide Institute) as a substrate and the reduced amount of thesubstrate can be calculated.

As mentioned above, the neutral metalloprotease according to theinvention purified from the recovered cells, in particular from thesurface of the cells, or from the supernatant of the culture, can beanalyzed for the N-terminal amino acid sequence by a gas phase proteinsequencer to determine the partial amino acid sequence. Furthermore, theenzymatic properties (optimum pH, pH stability, optimum temperature, theeffect of an inhibitor, etc.) of the isolated and purified neutralmetalloprotease can be examined.

In one embodiment of the present invention, the neutral metalloproteaseSVP35 was obtained from the surface of the cells of Streptoverticilliummobaraense and the neutral metalloprotease SVP70 can be obtained fromthe culture supernatant of Streptoverticillium mobaraense.

In one embodiment of the present invention, the neutral metalloproteaseaccording to the invention is the neutral metalloprotease SVP35 havingthe following properties:

1) Molecular weight: about 35,000 (as measured by SDS-PAGE)

2) Optimum pH: 6.0-8.0, more specifically 6.5-7.5, in particular around7.0

3) pH Stability: pH of 4-10

4) Optimum temperature: about 45° C.

5) Temperature stability: it is stable below about 50° C.

6) Inhibitors: it is strongly inhibited by ethylene diamine tetraaceticacid, 1,10-phenanthroline and phosphoramidon which are metalloproteaseinhibitors, and by Streptomyces subtilisin inhibitor (SSI) derived fromactinomycetes.

In another embodiment of the present invention, the neutralmetalloprotease according to the present invention is the neutralmetalloprotease SVP70 having the following properties:

1) Molecular weight: about 71,000 (as measured by SDS-PAGE)

2) Optimum pH: 6.0-8.0, more specifically 6.5-7.5, in particular around7.0

3) pH Stability: pH of 5-10

4) Optimum temperature: about 50° C.-55° C., in particular around 55° C.

5) Inhibitors: it is strongly inhibited by ethylene diamine tetraaceticacid, 1,10-phenanthroline and phosphoramidon which are metalloproteaseinhibitors, dithiothreitol which is a SH-reductant, and by Streptomycessubtilisin inhibitor (SSI) from actinomycetes.

When SVP35 or SVP70 is contacted with pro-MTG, both of them show highlyselective cleavage activity on the pro-structure part of the MTG.Namely, since both of the enzymes are characterized by converting thepro-MTG into the active MTG efficiently, while the activity fordegrading the resulting active MTG itself is low, both of them aresuitable enzymes for producing an active MTG using pro-MTG as a rawmaterial. The N-terminal amino acid sequences of the two new neutralmetalloproteases are shown in SEQ ID NO: 1 for SVP35, and in SEQ ID NO:2 for SVP70, which reveals the homology between these sequences.Therefore, sequences having any homology with these proteases in theirN-terminal amino acid sequences were searched and a metalloprotease SGMPII (J. Biochem. Vol. 110, p. 339-344 (1991)) from Streptomyces griseusas well as the three metalloproteases (GenBank/EMBL/DDBJ CAB76000,CAB76001, CAB69762) from Streptomyces coelicolor, and the like werefound. These proteases can also be used in the same manner as SVP35 andSVP70 for selective cleavage of the pro-structure part of a pro-MTG, andthey can be used to produce an active MTG using a pro-MTG as the rawmaterial.

Next, a method of producing the neutral metalloprotease according to thepresent invention by recombinant DNA technique will be described.

A number of examples of producing useful proteins including enzymes,physiologically active substances, and the like using recombinant DNAtechniques have been known. The advantage of using recombinant DNAtechniques is the ability to mass-produce useful proteins that exist insmall quantities in nature.

To produce the neutral metalloprotease according to the presentinvention by using recombinant DNA techniques, a genetic construct isgenerated first which contains a promoter, a sequence encoding a propersignal peptide, a nucleic acid fragment encoding the neutralmetalloprotease according to the invention, and a regulatory sequence(an operator or terminator, etc.) which is necessary to express the genefor the neutral metalloprotease in a coryneform bacterium, and properlypositioned so that they can function. The neutral metalloproteaseaccording to the invention may have a pro-structure part at theN-terminal. Vectors, which can be used for this construct, are notparticularly limited and include any one which can function in acoryneform bacterium, and may be those which autonomously replicate suchas plasmids or which are integrated into the chromosome of thebacterium. When a coryneform bacterium is used as a host, plasmidsderived from coryneform bacteria are particularly preferable as vectors.These include, for example, pHM1519 (Agric. Biol. Chem., 48, 2901-2903(1984)), pAM330 (Agric. Biol. Chem., 48, 2901-2903 (1984)), and modifiedplasmids, which possess drug-resistant genes.

Examples of Corynebacterium which can be used as a host bacterium in thepresent invention include mutant strains derived from wild-type strainsincluding Brevibacterium saccharolyticum ATCC14066, Brevibacteriumimmariophilum ATCC14068, Brevibacterium lactofermentum (Corynebacteriumglutamicum) ATCC13869, Brevibacterium roseum ATCC13825, Brevibacteriumflavum (Corynebacterium glutamicum) ATCC14067, Corynebacteriumacetoacidophilum ATCC13870, Corynebacterium glutamicum ATCC13032,Corynebacterium lilium (Corynebacterium glutamicum) ATCC15990,Brevibacterium ammoniagenes (Corynebacterium ammoniagenes) ATCC6871 andthe like, or mutant strains derived from mutants strain of thesewild-types.

Mutant strains which can be used in the present invention include, forexample, mutant strains defective in the ability to produce glutamate,mutant strains for amino acid production, such as lysine and the like,and mutant strains modified to produce other substances such as nucleicacids, for example, inosine. Such mutant strains can be obtained bytreatment with ultraviolet irradiation or a chemical mutagen such asN-methyl-N′-nitrosoguanidine and the like, and then selecting thestrains which have an increased ability to secreto-produce proteins.

Especially, Corynebacterium glutamicum AJ1203 (FERM BP-734) (originallydeposited on Mar. 26, 1984) (currently, National Institute of AdvancedIndustrial Science and Technology, International Patent OrganismDepositary, Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi,Ibaraki-ken, 305-8566, Japan), was isolated from the wild-typeCorynebacterium glutamicum (C. glutamicum) ATCC13869 as astreptomycin-resistant mutant strain. This strain is expected to have amutation in a functional gene associated with protein secretion, and itsability to secreto-produce heterologous proteins is extremelyhigh—approximately 2- or 3-fold as compared with the parent (wild-type)strain—under optimum culture conditions. Therefore, this strain issuitable as a host bacterium (see WO 02/081694). In addition, it ispreferable to use a strain which was obtained by modifying a host strainsuch that the strain no longer produces the cell surface protein,because the purification of the heterologous proteins secreted in themedium will be easier, so it is particularly preferable. Such amodification can be performed by introducing a mutation into the cellsurface protein gene on the chromosome or into its expression regulatoryregion through mutagenesis or gene recombination techniques.

Examples of promoters from a coryneform bacterium include promoters forthe genes of the cell surface proteins PS1, PS2, and SlpA, promoters forthe genes in biosynthetic systems of various amino acids, for example,glutamine synthetase gene, aspartokinase gene in the lysine biosyntheticsystem, and the like.

The signal peptide which is used in the present invention is the signalpeptide for a secretory protein from coryneform bacterium, the host, andpreferably it is the signal peptide of a cell surface protein from acoryneform bacterium. The cell surface proteins of coryneform bacteriainclude PS1 and PS2 from C. glutamicum (JP-Kokai No. 6-502548), and SlpAfrom C. ammoniagenes (JP-Kokai No. 10-108675).

To produce a neutral metalloprotease whose activity for selectivecleavage of a pro-structure of a pro-MTG is strong by using recombinantDNA techniques, a DNA encoding such a neutral metalloprotease isrequired.

In one embodiment of the present invention, the neutral metalloproteaseSVP35 is produced by using recombinant DNA techniques. The DNA encodingSVP35 can be obtained as follows.

First, the amino acid sequence of the purified SVP35 is determined. TheEdman method (Edman, P., Acta Chem. Scand. 4, 227 (1950)) can be used todetermine the amino acid sequence. The gas-phase protein sequencer fromShimadzu Co. Ltd. Co. Ltd. and the like can also be used to determinethe amino acid sequence.

For the neutral metalloprotease SVC35 according to the presentinvention, the sequence shown in SEQ ID NO: 1 has been found bysequencing 20 amino acid residues from the N-terminus.

This information can be used to synthesize an appropriate primer for PCRand generate a probe to obtain the neutral metalloprotease according tothe present invention. For example, a protease gene from actinomycetes,which is expected to have a homology based on the search results for thehomology in the N-terminal amino acid sequence, for example, ametalloprotease (GenBank/EMBL/DDBJ CAB76001) gene from Streptomycescoelicolor can be subjected to PCR using an actinomycetes DNA preparedby the method of Saito and Miura [Biochem. Biophys. Acta, 72, 619(1963)] as a template, to amplify the fragment of the gene encoding thisprotease. The amplified fragment can be used as a probe.

Then, the actinomycetes DNA prepared by the method of Saito and Miura,for example, the chromosomal DNA of Streptoverticillium mobaraenseIFO13819, is digested with appropriate different restriction enzymes,for example various restriction enzymes which recognize 6-basesequences. The digested actinomycetes chromosomal DNA can be analyzed bytechniques well known to those skilled in the art such as the Southernblot hybridization technique described in Molecular Cloning 2nd edition[J. Sambrook E. F. Fritsch and T. Maniatis, Cold Spring HarborLaboratory Press, p9. 31 (1989)] and the like by using the ³²P-labeledPCR-product obtained by the above-described PCR. For example, thenucleic acid molecule encoding the neutral metalloprotease according tothe present invention or the part thereof can be cloned by recoveringthe fragment which has been confirmed by Southern blot to have a highhomology with the chosen probe, and cloning it into an appropriatevector. The techniques necessary for such a gene cloning are well knownto those skilled in the art (see for example, J. Sambrook E. F. Fritschand T. Maniatis, Cold Spring Harbor Laboratory Press, p1. 90 (1989)).

In one embodiment of the present invention, PCR is performed using thechromosomal DNA of Streptomyces coelicolor A3(2) as a template toproduce a probe. Furthermore, a single band of about 8 kb which is ableto hybridize with ³²P-labeled probes detected in the digested product ofStreptoverticillium mobaraense IFO13819 chromosomal DNA digested withSphI. Thus, the chromosomal DNA of Streptoverticillium mobaraenseIFO13819 prepared by the foregoing method is digested with SphI, thefragment of about 8 kb is recovered through an agarose gelelectrophoresis, the recovered fragment is introduced at the SphI sitein pUC18, and then it is introduced into a competent cell of Escherichiacoli JM109 to generate a library. The clones of interest can be obtainedby screening the generated library using a synthetic oligonucleotide asa probe according to colony hybridization techniques described inMolecular Cloning 2nd edition (supra), and selecting the strain whichharbors the plasmid containing the gene fragment of SVP35 cloned intothe plasmid. The plasmid recovered from this strain is herein designatedpVSV1. The nucleotide sequence of the fragment cloned into pVSV1 isanalyzed, the primary amino acid sequence is deduced to confirm that thefragment encodes the previously determined N-terminal amino acidsequence. Thus, the obtained gene is confirmed to be the gene encodingSVP35.

Then, a recombinant nucleic acid molecule can be constructed to expressthe neutral metalloprotease according to the present invention byligating a genetic construct containing the DNA encoding the obtainedmetalloprotease to an appropriate vector depending on the properties ofthe chosen host. The host coryneform bacterium cells are transformedwith the recombinant nucleic acid molecule. The transformed cells can becultured in a suitable medium to recover the neutral metalloproteaseaccording to the present invention which is secreted and/or accumulatesin the medium and/or in the cell.

Next, a method of producing an active MTG from pro-MTG using the neutralmetalloprotease will be described.

The neutral metalloprotease used in the production of an active MTG canbe reacted with a pro-MTG as a fraction containing the neutralmetalloprotease prepared from the culture medium of a neutralmetalloprotease producing bacterium. It can be also used as a morehighly purified neutral metalloprotease with high specific activity.Furthermore, as described below, the neutral metalloprotease can be alsoused, wherein the neutral metalloprotease can be obtained by culturingthe cell transformed with a recombinant nucleic acid molecule which maybe obtained by connecting a DNA encoding a neutral metalloproteasehaving strong selective cleavage activity for a pro-structure part ofpro-MTG.

The pro-MTG used to produce MTG may be a fraction containing the pro-MTGprepared from the culture medium a pro-MTG producing bacterium. Morehighly purified pro-MTG may also be used. The reaction may be performedunder conditions such that the amount of a neutral metalloprotease addedto the pro-MTG is from 1/10 to 1/500 by weight and is appropriatelyadjusted within the reaction temperature of between 15° C. and 50° C.and the pH range of between pH 5.0 and 9.

In addition, the genetic construct, which is constructed as describedabove and which contains the DNA encoding the neutral metalloproteaseaccording to the present invention, may be introduced into amicroorganism containing the genetic construct encoding a pro-MTG, inparticular, into a coryneform bacterium, to produce in a singlebacterial cell both the pro-MTG and the neutral metalloproteaseaccording to the present invention, thereby the pro-MTG may be convertedinto a mature MTG under the above conditions. A more detailed method forefficiently producing a pro-MTG in coryneform cells, the geneticconstruct used for such a method, and a coryneform bacterium into whichthe genetic construct has been introduced are disclosed in, for exampleWO 01/23591. More specifically, for example, a coryneform bacteriumwhich can efficiently secrete a pro-MTG protein extracellularly may beobtained by introducing a genetic construct into a coryneform bacterium,wherein the genetic construct is obtained by connecting the sequenceencoding a pro-MTG, which is located downstream of the sequence encodingthe signal peptide domain of a coryneform bacterium, particularly thesignal peptide domain of a cell surface protein, downstream of anappropriate promoter. The signal peptide, promoter, and host which canbe used for this purpose can be selected from signal peptides,promoters, and hosts which are suitable for expressing the neutralmetalloproteases according to the present invention and as mentionedabove. A combination of vectors that are compatible in the same cell isalso well known to those skilled in the art. Therefore, the mature MTGcan be obtained by introducing an appropriate genetic expressionconstruct containing the DNA encoding the neutral metalloproteaseaccording to the present invention as mentioned above into a coryneformbacterium producing a pro-MTG, or vice versa, by introducing anappropriate genetic expression construct encoding a pro-MTG intocoryneform bacterium producing the neutral metalloprotease according tothe present invention, thereby allowing the genetic constructs which canexpress the pro-MTG and the neutral metalloprotease according to thepresent invention to coexist in the same bacterium, culturing thebacterium, and maintaining the culture under appropriate conditions suchthat the neutral metalloprotease according to the present invention isactive.

The transglutaminase produced by the method according to the presentmethod can be isolated and purified from the reaction mixture accordingto methods well known to those skilled in the art. For example, thetransglutaminase can be isolated and purified by removing the cells fromthe mixture by centrifugation, etc. and then by using known appropriatemethods such as salting-out, ethanol precipitation, ultrafiltration, gelfiltration chromatography, ion-exchange column chromatography, affinitychromatography, medium high-pressure liquid chromatography,reversed-phase chromatography, hydrophobic chromatography, or acombination thereof.

The present invention is further described in the following non-limitingExamples.

EXAMPLES Example 1 Neutral Metalloprotease Produced byStreptoverticillium mobaraense IFO13819

(1) Purification of Neutral Metalloprotease (SVP70) Produced byStreptoverticillium mobaraense IFO13819

800 mL of ISP2 culture medium (4 g of Yeast Extract, 10 g of MaltExtract, 4 g of Glucose per liter of water, adjusted to pH 7.3) wasplaced in a 5 L Sakaguchi flask (shaking flask), and was inoculated withStreptoverticillium mobaraense IFO13819 from a plate, and cultured byshaking at 30° C. for 9 days at 120 rpm. The culture medium wascentrifuged, and the supernatant of the culture was collected. It wasfiltered using a Depth filter (3 μm of pore size, Sartorius Co. Ltd.),followed by concentration using Sartocon Slice membrane having a poresize of 10,000 Da (Saltorius Co. Ltd.). The concentrate was diluted10-fold with Tris-HCl buffer/5 mM calcium chloride (pH 7.5), subjectedto a DEAE-Sepharose FF (2.6φ×10 cm, Amersham Pharmacia Co. Ltd.) columnequilibrated with the same buffer, using FPLC (Amersham Pharmacia Co.Ltd.), and eluted using a linear concentration gradient of 0-0.5 Msodium chloride. A fraction containing the active ingredient wascollected, subjected to a phenyl Sepharose HP (1.6φ×10 cm, AmershamPharmacia Co. Ltd.) column equilibrated with 1.5 M ammonium sulfate/20mM MES buffer/5 mM calcium chloride (pH 6.0), eluted using linearconcentration gradient of 1.5-0 M ammonium sulfate, and an activefraction was collected. The resulting active fraction was dialyzedagainst 20 mM MES buffer/5 mM calcium chloride (pH 6.0) at 4° C.overnight to obtain a purified enzyme solution.

The measurement of the enzyme activity at each step was carried asfollows:

The enzyme solution was added to 20 mM sodium phosphate buffercontaining peptide GPSFRAPDS (Peptide Institute) (SEQ ID NO: 11) toyield 170 μl of total liquid volume, and it was reacted at 30° C. for 10minutes, followed by heating at 95° C. for 5 minutes to terminate thereaction. 80 μl of this solution was analyzed by HPLC under thefollowing conditions and its activity was calculated based on thedecreased amount of the substrate.

Apparatus: HPLC L-6300 system (Hitachi Co. Ltd).

Column: YMC-PACK ODS 120A 4.6×150 mm (YMC)

Eluent: (A) 0.1% TFA (B) 80% acetonitrile/0.1% TFA

Eluting condition: linear concentration gradient of 12-16% acetonitrile(for 15 minutes)

Flow rate: 1.0 ml/min

Detection wavelength: 220 nm

Under these conditions, the peptide GPSFRAPDS (SEQ ID NO: 11) was elutedfor 13 to 14 minutes of retention time, and the degraded product FRAPDS(SEQ ID NO: 12) was eluted for 7.5 to 8.5 minutes of retention time.

The amount of the enzyme that catalyzes one (1) nmol of pro-MTGdegradation in a minute was defined as one (1) unit of the enzymeactivity.

(2) Purification of Neutral Metalloprotease (SVP35) Produced byStreptoverticillium mobaraense IFO13819

800 mL of ISP2 culture medium was placed in a 5 L of Sakaguchi flask andwas inoculated with Streptoverticillium mobaraense IFO13819 from aplate, and cultured by shaking at 30° C. for 48 hours at 120 rpm. Theculture medium was centrifuged, and the supernatant of the culture wasdiscarded to harvest cells. The cells were suspended in 20 mM Tris-HClbuffer/30 mM sodium chloride (pH 7.5), shaken on ice for 4 hours, andthen the supernatant was collected by centrifugation. The supernatantobtained was filtered and sterilized using a Depth filter (0.22 μm ofpore size, made by Sartorius Co. Ltd.), and then it was subjected to aCM-Sepharose FF (Amersham Pharmacia Co. Ltd.) column (1.6φ×10 cm)equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM calciumchloride and 0.01 mM zinc chloride, using FPLC (Amersham Pharmacia Co.Ltd.), eluted in the same buffer using a linear concentration gradientof 0-0.5 M sodium chloride. A fraction containing the active ingredientwas collected, and was further subjected to Phenyl-Sepharose HP column(1 mL, Amersham Pharmacia Co. Ltd.) equilibrated with 20 mM Tris-HClbuffer containing 1.5 M ammonium sulfate, 5 mM calcium chloride and 0.01mM zinc chloride, and eluted using a linear concentration gradient of1.5-0 M ammonium sulfate. An active fraction was collected, anddemineralized by 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM calciumchloride and 0.01 mM zinc chloride, using PD-10 column (AmershamPharmacia) to give a partially purified enzyme solution.

The enzyme activity at each step was measured using the peptideGPSFRAPDS as a substrate in the same manner as in (1).

(3) Evaluation of the Properties of the Neutral Metalloprotease (SVP35)Produced by Streptoverticillium mobaraense IFO13819

i) Substrate Specificity

1 mg/ml of insulin B solution and pro-MTG solution prepared in 20 mMTris-HCl buffer (pH 7.5) containing 5 mM calcium chloride and 0.01 mMzinc chloride was used as a substrate, and an enzyme solution was addedto the solution to react at 30° C. for 2 hours, and then peptidefragments were separated by HPLC under the following conditions:

Apparatus: L-7100/7200/7405/D-7600 (Hitachi Co. Ltd.)

Column: VYDAC C18 4.6 mm I.D.×250 mm (VYDAC)

Eluent: (A) 0.1% TFA (B) 80% acetonitrile/0.1% TFA

Eluting condition: linear concentration gradient of 4-44% acetonitrile

Flow rate: 0.5 ml min

Detection wavelength: UV 220 nm

The amino acid sequences of the obtained peptide fragments were analyzedby PPSQ-10 (Shimadzu Co. Ltd.) to characterize the sequences of thecleavage points for SVP35. As a result, it was confirmed that thepeptide was cleaved before (at the N-terminal of) especially Phe, oftenLeu, sometimes Tyr, Trp, Ile, Val, and that SVP recognized the aromaticamino acids and hydrophobic amino acids with bulky side-chainspositioned at P′ 1 of the cleavage site.

ii) Optimum pH

In 0.15 M GTA buffer (buffered by 3,3-dimethyl glutaric acid, Tris(hydroxy methyl) amino methane, 2-amino-2-methyl-1,3-propanediol) frompH 3 to pH 10, SVP35 was allowed to act onGly-Pro-Ser-Phe-Arg-Ala-Pro-Asp-Ser (SEQ ID NO: 11) as a substrate at30° C. for 10 minutes. As a result, it was revealed that the Optimum pHof SVP35 was around 7.0, and that when the activity at pH 7.0 wasdefined as 100%, SVP35 had an activity of 70% or more at pH 6.0-8.0 andan activity of 80% or more at pH 6.5-7.5 (see FIG. 1).

iii) pH Stability

To 10 μl of SVP35 purified enzyme solution, 40 μl of each pH of 0.15 MGTA buffer from pH 3 to pH 10 was added, left at 4° C. overnight,followed by addition of 0.1 M sodium phosphate buffer (pH 7.0) to theliquid volume of 400 μl, and was adjusted to pH 7.0. To these enzymesolutions, Gly-Pro-Ser-Phe-Arg-Ala-Pro-Asp-Ser (SEQ ID NO: 11) was addedas a substrate, and reacted at pH 7.0, at 30° C. for 10 minutes. As aresult, it was shown that SVP35 was stable within the range of pH 4 topH 10 (when the activity at pH 4.0 was defined as 10000, it had anactivity of 90% or more at pH 4-10) (see FIG. 2).

iv) Optimum Temperature

To the purified enzyme solution diluted with 20 mM Tris-HCl buffer (pH7.5) containing 5 mM calcium chloride and 0.01 mM zinc chlorideGly-Pro-Ser-Phe-Arg-Ala-Pro-Asp-Ser (SEQ ID NO: 11) was added andreacted at pH 7.0, between 5° C. and 65° C. for 10 minutes. As a result,it was shown that the Optimum temperature of SVP35 was about 45° C. andit had high activity within the range of 40° C. to 50° C. (it had anactivity of 80% or more than that at 45° C.) (see FIG. 3).

v) Temperature Stability

To 10 μl of the purified enzyme solution, 40 μl of 20 mM Tris-HCl buffer(pH 7.5) containing 5 mM calcium chloride and 0.01 mM zinc chloride wasadded to treat at 4° C. or at from 30° C. to 70° C. for 15 minutes, andthen cooled by ice, added 250 μl of 20 mM sodium phosphate buffer(pH-7.0). To this enzyme solution, Gly-Pro-Ser-Phe-Arg-Ala-Pro-Asp-Ser(SEQ ID NO: 11) was added as a substrate and reacted at 30° C. for 5minutes. When the activity treated at 4° C. was defined as 100%, theremaining activity at each temperature was calculated. As a result, itwas shown that SVP35 retained 80% of activity at 50° C., but it lost itsactivity at 60° C. (see FIG. 4).

vi) Inhibitors

To 20 mM sodium phosphate buffer (pH 7.0) containing various compoundsat the concentrations shown in FIG. 5, the purified enzyme solution wasadded and left for 60 minutes at room temperature. ThenGly-Pro-Ser-Phe-Arg-Ala-Pro-Asp-Ser (SEQ ID NO: 11 was added as asubstrate and reacted for 10 minute at 30° C. The relative activity byadding each compound was calculated based on theGly-Pro-Ser-Phe-Arg-Ala-Pro-Asp-Ser (SEQ ID NO: 11) cleavage activity inthe absence of compounds as 100%. As a result, it was shown that SVP35was strongly inhibited by ethylene diamine tetraacetic acid,1,10-phenanthroline and phosphoramidon which are metalloproteaseinhibitors, and by Streptomyces subtilisin inhibitor (SSI) derived fromactinomycetes (see FIG. 5).

(4) Characterization of the Properties of the Neutral Metalloprotease(SVP70) Produced by Streptoverticillium mobaraense IFO13819

i) Substrate Specificity

The substrate specificity was examined similarly as described in (3)-i).As a result, it was revealed that the substrate was cleaved before (atN-terminal side of) especially Phe, often Leu, sometimes Tyr, Trp, Ile,Val, and that SVP70 recognized the aromatic amino acids and hydrophobicamino acids with bulky side-chains positioned at P′ 1 of the cleavagesite.

ii) Optimum pH

The Optimum pH of SVP70 was examined similarly as (3)-ii). As a result,it was revealed that the Optimum pH of SVP70 was around 7.0, and that ifthe activity at pH 7.0 is defined as 100%, SVP70 had an activity of 90%or more at pH 6.0-8.0 and an activity of 95% or more at pH 6.5-7.5 (seeFIG. 1).

iii) pH Stability

The pH stability was examined similarly to (3)-iii). As a result, it wasshown that SVP70 was stable within pH 5 to pH 10, but it was less stablethan SVP35 at slightly alkaline (see FIG. 2). Specifically, if theactivity at pH 5.0 was defined as 100%, it had an activity 90% or morein the range of pH 5 to pH 7, and it had an activity about 80% or moreeven in the range of pH 7 to pH 10.

iv) Optimum Temperature

The Optimum temperature of SVP70 was examined similarly as (3)-iv). As aresult, it was shown that the Optimum temperature of SVP70 was withinthe range from about 50° C. to 55° C., especially around 55° C. (seeFIG. 3).

v) Inhibitors

The inhibitory activities of various compounds to SVP70 were examinedanalogously to (3)-vi). As a result, SVP70 underwent strong inhibitoryaction by ethylene diamine tetraacetic acid, 1,10-phenanthroline, andphosphoramidon, which are metalloprotease inhibitors, and by reductantdithiothreitol, urea, and Streptomyces subtilisin inhibitor (SSI)derived from actinomycetes (see FIG. 5).

(5) Sequencing of the N-terminal Amino Acid Sequence of SVP35 and SVP70

The purified enzymes of SVP35 and SVP70 obtained in (1) and (2) abovewere transferred onto Polyvinilidene-difluoride (PVDF) membrane usingMembrane Cartridge (Perkin Elmer Co. Ltd.) and the N-terminal amino acidsequence was analyzed using a gas-phase Protein Sequencer PPSQ-10(Shimadzu Co. Ltd.). The amino acid sequence of SVP35 is shown in SEQ IDNO: 1, and the amino acid sequence of SVP70 is shown in SEQ ID NO: 2. Ahomology can be seen in these sequences.

Accordingly, those which had any homology to these proteases forN-terminal amino acid sequences were searched, and then metalloproteaseSGMP II (J. Biochem., Vol. 110, p. 339-344, 1991) from Streptomycesgriseus, and three metalloproteases (GenBank/EMBL/DDBJ CAB76000, thesame CAB76001, and the same CAB69762), etc. from Streptomyces coelicolorwere found. These proteases also can be used to cleave selectively thepro-structure part of the pro-MTG, and therefore they can be used toproduce an active MTG according to the present invention.

(6) Cloning of SVP35 Gene and Its Secretory Expression in CoryneformBacteria

The chromosomal DNA of Streptomyces coelicolor A3(2) was prepared usingthe method of Saito and Miura [Biochem. Biohhys. Acta, 72, 619 (1963)].Primers shown in SEQ ID NO: 3 and SEQ ID NO: 4 were synthesized byreferring to the sequence of metalloprotease (GenBank/EMBL/DDBJCAB76001) gene from Streptomyces coelicolor which have a homology in theN-terminal amino acid sequence. Primers shown in SEQ ID NO: 3 and SEQ IDNO: 4 were used to perform PCR using the chromosomal DNA of Streptomycescoelicolor A3(2) as a template, and the gene region in themetalloprotease gene was amplified. For the PCR reaction, Pyrobest DNApolymerase (Takarasyuzo Co. LTD.) was used and the reaction conditionsfollowed the protocol recommended by the manufacturer. The chromosomalDNA of Streptoverticillium mobaraense IFO13819 prepared by the method ofSaito and Miura was digested by various restriction enzymes whichrecognize 6-base sequence, the digested samples were analyzed by theSouthern blot hybridization as described in Molecular Cloning 2ndedition [J. Sambrook E. F. Fritsch and T. Maniatis, Cold Spring HarborLaboratory Press, p9. 31 (1989)], using the ³²P-labeled PCR product as aprobe, and a single band of about 8 kb was detected by SphI cleavage.Accordingly, the chromosomal DNA of Streptoverticillium mobaraenseIFO13819 which had been prepared by the foregoing method was digestedwith SphI, and a fragment of about 8 kb was recovered through agarosegel electrophoresis using EASYTRAP Ver. 2 (Takarasyuzo Co. LTD.). Therecovered fragment was inserted into SphI site of pUC18, which wasintroduced into competent cells of Escherichia coli JM109 (TakarasyuzoCo. LTD.) to generate a library. The library was screened for thebacterial strain which contains the plasmid where the SVP35 genefragment was cloned, by colony hybridization as described in MolecularCloning 2nd edition [J. Sambrook E. F. Fritsch and T. Maniatis, ColdSpring Harbor Laboratory Press, p1. 90 (1989)], using the syntheticnucleotide as a probe.

The plasmid was recovered from the strain obtained above and wasdesignated as pVSV1. The nucleotide sequence of the fragment cloned inpVSV 1 was determined. The nucleotide sequence of this cloned fragmentis shown in SEQ ID NO: 5. The primary amino acid sequence encoded bythis gene was deduced, which allowed the determination of the entireprimary amino acid sequence of SVP35 containing the signal sequence ofSVP35 including the amino acid sequence of the previously determinedN-terminal portion and the region assumed to be a pro-structure part.The entire amino acid sequence of SVP35 is shown in SEQ ID NO: 6. It ispresumed that amino acids nos. 1-36 of amino acid sequence described inSEQ ID NO: 6 refer to the signal sequence, amino acids nos. 37-216 referto the pro-structure part, and amino acids nos. 217-537 correspond tothe mature SVP35.

Primers shown in SEQ ID NO: 7 and SEQ ID NO: 8 were synthesized usingpVSV1 as a template by referring to the sequence of SEQ ID NO: 5, andthe gene region containing the pro-structure part of SVP35 and themature SVP35 was amplified by PCR. For the PCR reaction, Pyrobest DNApolymerase (Takarasyuzo Co. Ltd.) was used and the reaction conditionsfollowed the protocol recommended by the manufacturer.

Next, using pPKSPTG1 described in WO 01/23591 as a template, the regionincluding the 5′-upstream region containing the promoter region of PS2gene which is the cell surface protein of C. glutamicum and the signalsequence of SlpA, the cell surface protein of C. ammoniagenes wasamplified by PCR technique using the combination of oligonucleotides ofSEQ ID NO: 9 and SEQ ID NO: 10. The primer shown in SEQ ID NO: 10contains the sequence encoding the N-terminal amino acids of SVP35having a pro-structure part.

Then, the gene of the heterologous fusion pre-pro SVP35 gene fragment,which was connected to the 5′-upstream region comprising the promoterregion of PS2 gene and the signal sequence of SlpA, the cell surfaceprotein, from C. ammoniagenes, was amplified by performing cross-overPCR with SEQ ID NO: 8 and SEQ ID NO: 9 using the mixture of 1 μl each ofthe amplified PCR solution. The amplified fragment of about 2.3 kb wasdetected by agarose gel electrophoresis. The PCR product was subjectedto agarose gel electrophoresis to recover a fragment of about 2.3 kb,and after blunting its ends using DNA Blunting Kit (Takarasyuzo Co.Ltd.), the fragment was inserted into SmaI site of pCV7 as described inJP-Kokai No. 9-070291 to obtain pVSV1. The nucleotide sequence of theinserted fragment was determined according to a conventional method toconfirm that the fusion gene was constructed as expected.

C. glutamicum ATCC13869 was transformed with the constructed pVSV1 andthe strains which grew on the CM2S agar medium containing 5 mg/l ofchloramphenicol (10 g of yeast extract, 10 g of tryptone, 5 g ofsucrose, 5 g of NaCl, 15 g of agar per liter of distilled water) wereselected. Then, the selected C. glutamicum ATCC13869 harboring pVSV1 wascultured in MMTG culture medium (60 g of glucose, 0.4 g of magnesiumsulfate heptahydrate, 30 g of ammonium sulfate, 1 g of potassiumdihydrogenphosphate, 0.01 g of ferrous sulfate heptahydrate, 0.01 g ofmanganese sulfate pentahydrate, 450 μg of thiamine hydrochloride, 450 μgof biotin, 0.15 g of DL-methionine, 50 g of calcium carbonate per literof distilled water, adjusted to pH 7.5) containing 5 mg/l ofchloramphenicol at 30° C. for 30 hours. 1 ml of the culture medium wascentrifuged to separate to the supernatant of the culture and thebacteria. The activity of SVP35 was detected in the supernatant of theculture, and as a result of SDS-PAGE (Nature, 227, 380-685 (1970))electrophoresis according to Laemmli's method, it was confirmed thatabout 200 mg/L of SVP35 was secretory-expressed.

Example 2 Conversion of Transglutaminase from Streptoverticilliummobaraense IFO13819 (pro-MTG) into an Active Form

Using pro-MTG (1 mg/ml) expressed by Corynebacterium glutamicum as apurified substrate, the neutral protease (SVP35, SVP70) fromStreptoverticillium mobaraense or the neutral metalloprotease SGMP IIfrom Streptomyces griseus was mixed in the ratio of the substrate: theenzyme=200:1, the mixture was reacted at 30° C. After 0, 1, 2, 4, 7, 20hours, the reaction mixture was sequentially picked up, and the aliquotsof the reaction mixture were mixed with SDS-PAGE sample buffer andheated at 95° C. for 3 minutes, and then subjected to SDS-PAGE accordingto Laemmli's method (Nature, 227, 680-685 (1970)). The result is shownin FIG. 6. As can be seen in FIG. 6, when these proteases were reacted,pro-MTGs were converted to the mature forms, and the produced MTGs werenot reduced even after a long-term reaction. The transglutaminase (TG)activity of the picked up fraction was measured by the hydroxamatemethod, and sufficient activity was confirmed. In addition, SGMP II waspurified from actinase (Kakenseiyaku Co. Ltd.) according to thereference method (J. Biocem., Vol. 110, p. 339-344, 1991).

Then, the neutral metalloprotease SVP70 from Streptoverticilliummobaraense, and serine protease SAM-P45 (Streptomyces albogriseolus) asa control, were added to the pro-MTGs with gradually increasing amountsof these enzymes, and reacted at 30° C. and pH 7.0. After 1, 4, 7, and24 hours, the reaction mixture was picked up sequentially to determinethe TG activity by the hydroxamate method (see FIG. 7). The proteinconcentration of TG was measured by reverse phase chromatography (seeFIG. 8). As a result, it was shown that SVP could convert pro-MTG toactive MTG with an amount as small as 1/500 of the substrate. It wasshown that SAM-P45 generated only insufficient transglutaminase activityeven at an amount of 1/50 of the substrate, and that the completeconversion to the active form was not observed. On the other hand, whenSAM-P45 was added at an amount of 1/10 of the substrate, the conversioninto the active MTG was observed, but a decrease in the amount and theactivity of MTG-protein was observed. This suggests thatover-degradation of the mature MTG occurred by SAM-P45.

The present invention provides a new protease from an actinomycetes,Streptoverticillium mobaraense, which specifically cleaves thepro-structure part of transglutaminase precursor to activate it, and thegene thereof. The new protease according to the present invention can beexpressed in a large amount by a coryneform bacterium, and thereby thepresent invention provides a method for efficiently producingtransglutaminase from microorganisms.

The advantage of using the neutral metalloproteases from actinomycetesaccording to the present invention for the production of an active MTGis that these enzymes have strong activities for selectively cleavingthe pro-structure part of the pro-MTG, and that these enzymes can beexpressed extracellularly by a coryneform bacterium.

As it is shown that the pro-MTG from actinomycetes can be efficientlyexpressed and secreted by a coryneform bacterium, it is possible toproduce more efficiently an active MTG by a single bacterial cell byco-expressing and secreting the pro-MTG and the neutral metalloprotease.In this instance, it is sufficient to express the neutralmetalloprotease in only an amount required and sufficient for cleavingthe pro-structure part of the pro-MTG.

While the invention has been described in detail with reference topreferred embodiments thereof, it will be apparent to one skilled in theart that various changes can be made, and equivalents employed, withoutdeparting from the scope of the invention. Each of the aforementioneddocuments, including the foreign priority document, JP 2003-061623, isincorporated by reference herein in its entirety.

REFERENCES

-   1. JP-Kokoku No. 1-50382-   2. JP-Kokai No. 64-27471-   3. WO publication No. 01/2351-   4. JP-Kokai No. 6-502548-   5. JP-Kokai No. 10-108675-   6. Eur. J. Biochem., Vol. 257, pages 570-576, 1998,-   7. J. Biochem., Vol. 110, pages 339-344, 1991.

1. A biochemical or cellular method of producing an active Streptoverticillium transglutaminase from a Streptoverticillium protransglutaminase comprising contacting a neutral Streptoverticillium mobaraense IFO13819 metalloprotease with the protransglutaminase, wherein said metalloprotease is produced by culturing a microorganism into which the gene encoding the metalloprotease has been introduced, and recovering the active transglutaminase, wherein said metalloprotease comprises: a molecular weight of about 35,000, an optimum pH 6.5 to pH 7.5, an optimum temperature of about 45° C., and an activity to cleave the peptide of SEQ ID NO: 11 wherein, said metalloprotease is strongly inhibited by ethylene diamine tetraacetic acid, 1,10-phenanthroline, phosphoramidon, and Streptomyces subtilisin inhibitor (SSI) from actinomycetes.
 2. The method according to claim 1, wherein said microorganism is a coryneform bacterium.
 3. The method according to claim 1, wherein the metalloprotease comprises, as an N-terminal amino acid sequence, the sequence of SEQ ID NO:
 1. 4. The method according to claim 1, wherein the metalloprotease comprises the amino acid sequence of amino acids 217-537 of SEQ ID NO:6.
 5. The method of claim 1, wherein a gene encoding the protransglutaminase has been introduced into the microorganism, and said protransglutaminase is produced by culturing the microorganism.
 6. The method of claim 2, wherein a gene encoding the protransglutaminase has been introduced into the microorganism, and said protransglutaminase is produced by culturing the microorganism.
 7. The method of claim 3, wherein a gene encoding the protransglutaminase has been introduced into the microorganism, and said protransglutaminase is produced by culturing the microorganism.
 8. The method of claim 4, wherein a gene encoding the protransglutaminase has been introduced into the microorganism, and said protransglutaminase is produced by culturing the microorganism. 